Plant acyl-CoA-binding proteins (ACBPs) function in plant development and stress responses, with some ACBPs interacting with protein partners. This study tested the interaction between two Class II GmACBPs (Glycine max ACBPs) and seven kinases, using yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC). The results revealed that both GmACBP3.1 and GmACBP4.1 interact with two soybean kinases, a mitogen-activated protein kinase MPK2, and a serine/threonine-protein kinase SAPK2, highlighting the significance of the ankyrin-repeat (ANK) domain in facilitating protein-protein interactions. Moreover, an in vitro kinase assay and subsequent Phos-tag SDS-PAGE determined that GmMPK2 and GmSAPK2 possess the ability to phosphorylate Class II GmACBPs. Additionally, the kinase-specific phosphosites for Class II GmACBPs were predicted using databases. The HDOCK server was also utilized to predict the binding models of Class II GmACBPs with these two kinases, and the results indicated that the affected residues were located in the ANK region of Class II GmACBPs in both docking models, aligning with the findings of the Y2H and BiFC experiments. This is the first report describing the interaction between Class II GmACBPs and kinases, suggesting that Class II GmACBPs have potential as phospho-proteins that impact signaling pathways.
Keywords: bimolecular fluorescence complementation; kinases; phosphorylation; plant acyl-CoA-binding proteins; yeast two-hybrid.